PROJECT SUK/IH/IARY (See instructions): The progressive growth of primary ovarian cancer and metastasis is dependent on development of an adequate blood supply (angiogenesis). Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis and consequent ovarian cancer growrth and progression. VEGF blockade has shown promise in human studies. Our pre-clinical and clinical results from the prior funding period demonstrate that a novel approach (high-affinity VEGF decoy receptor, VEGF-Trap or aflibercept) for VEGF blockade was highly effective in combination with taxane chemotherapy. However, despite initial responses, most patients eventually develop tumor progression resulting in their demise, mainly due to the development of drug resistance. The DII4 (delta-like ligand 4)/Notch signaling pathway has recently been shown to play an important role in angiogenesis including vessel maturation, pericyte recruitment, branching and cell differentiation, proliferation, survival and apoptosis. Our preliminary data indicate that when DII4 inhibition using a monoclonal antibody (DII4-mAb or REGN421) was coupled with VEGF inhibition (aflibercept), this combination strikingly reduced tumor burden and ascites, suggesting that this anti-angiogenesis regimen holds promise as a novel therapeutic modality. However, the mechanisms of its potency are not fully understood. We hypothesize that the DII4/Notch signaling pathway plays an important role in reducing the efficacy of anti-VEGF monotherapy and targeting both VEGF and DII4/Notch signaling pathway will enhance anti-angiogenic therapy. The overall goal of this proposal is to develop novel and effective strategies for targeting ovarian cancer angiogenesis. In the current proposal, we will investigate the functional significance of DII4/Notch signaling pathway in ovarian cancer by examining whether DII4 expression in ovarian cancer cells activates Notch signaling in endothelial cells and whether blockade of DII4/Notch signaling with REGN421 deregulates angiogenesis in vitro in Aim 1. In Aim 2, we will assess the efficacy of combinatorial approaches for targeting VEGFA/EGFR/DII4/Notch signaling pathway with aflibercept and REGN421 using in vivo orthotopic ovarian cancer models. We will conduct a Phase I/lb clinical trial using the anti-DII4 monoclonal antibody REGN421 in patients with recurrent ovarian carcinoma in Aim 3. Thus, all three Aims are complementary to each other and findings of this study should allow the design of new therapeutic approaches for women with ovarian cancer.